Vision loss may be caused by disease or damage to the retina of the eye. The retina consists of a specialized layer of cells at the back of the eye where light entering the eye is sensed as an image. These cells normally respond to all aspects of the light emitted from an object and allow perception of color, shape and intensity. The types of cells located in the retina include retinal pigment epithelial ("RPE") cells, rod cells, cone cells, bipolar cells, amacrine cells, horizontal cells, Mueller cells, glial cells, and retinal ganglion cells.
When normal retinal function is impaired, it may lead to a loss of color perception, blind spots, reduced peripheral vision, night blindness, photophobia, decreased visual acuity or blindness. For example, acquired immunodeficiency virus ("AIDS") patients may suffer cytomegalovirus retinitis which is caused by spread of the cytomegalo virus to the retina (Bloom et al., Medicine, 109(12): 963-968 (1988)). This and other infectious processes can lead to loss of visual field, decreased visual acuity, and blindness.
Uveitis is an inflammation of the eye which can affect the retina and can lead to decreased visual acuity. Its effects on the retina include inflamed or leaking vasculature which may appear as perivascular exudation or haemorrhage, oedema of the retina, chorioretinal lesions, neovascularization or inflammatory changes in the peripheral retina. (Anglade et al., Drugs, 49(2):213-223 (1995)).
Cancers of the retina also impair vision. One example is retinoblastoma, which is a childhood type of cancer. Other diseases may occur through age-related macular degeneration.
Many different genetic diseases lead to retinal damage and blindness. A relatively common example is retinitis pigmentosa ("RP"), which affects one person in four thousand worldwide. Patients with RP have normal vision for one or more decades, and then experience progressive loss of vision due to the premature death of rod or cone cells. Blindness may result. Other types of retinal degenerations (retinal dystrophies) may result from the programmed death of other retinal cell types.
Physical damage to retinal cells may also occur through retinal detachment which leads to retinal degeneration and blindness.
The therapeutic strategies for treating loss of vision caused by retinal cell damage vary, but they are all directed to controlling the illness causing the damage, rather than reversing the damage caused by an illness by restoring or regenerating retinal cells. As one example, the treatments of uveitis are drawn from the knowledge of changes in the retinal environment when inflammation occurs. Corticosteroids, such as prednisone, are the preferred drug of treatment. However, these drugs are immunosuppressants with numerous side effects. As well, the systemic immunosuppression may have significant negative effects on the development of children as well as on adults in poor health such as the elderly and patients with chronic disease. These patients must try alternate drugs such as alkylating agents or antimetabolites which also have side effects. Clearly, patients with eye diseases remain vulnerable to sustaining permanent damage to the retinal cells, even if drug treatments are available.
There are no known successful treatments for RP and other retinal dystrophies. There are also no treatments which regenerate new cells endogenously or which transplant healthy tissue to the retina. Even if it were possible to develop some form of transplantation, it would be subject to the same problems that accompany transplants in other organ systems. These include:
in many cases, implants provide only temporary relief as the symptoms associated with the disease often return after a number of years, PA1 rejection by the patient of foreign tissue, PA1 adverse reactions associated with immunosuppression (immunosuppression is needed to try to help the patient accept the foreign tissue), PA1 the inability of a sufficient number of cells in the tissue being implanted to survive during and after implantation, PA1 transmitting other diseases or disorders may be transmitted to the patient via the implant, and PA1 the results may not justify the costs and efforts of a complex procedure. PA1 Proliferate: Stem cells are capable of dividing to produce daughter cells. PA1 Exhibit self-maintenance or renewal over the lifetime of the organism: Stem cells are capable of reproducing by dividing symmetrically or asymmetrically. Symmetric division is a source of renewal of stem cells. Symmetric division leads to increases in the number of stem cells. Asymmetric division maintains a consistent level of stem cells in an embryo or adult mammal. PA1 Generate large number of progeny: Stem cells may produce a large number of progeny through the transient amplification of a population of progenitor cells. PA1 Retain their multilineage potential over time: Stem cells are the ultimate source of differentiated tissue cells, so it is a characteristic that they retain their ability to produce multiple types of progenitor cells, which will in turn develop into specialized tissue cells. PA1 Generate new cells in response to injury or disease: This is essential in tissues which have a high turnover rate or which are more likely to be subject to injury or disease, such as the epithelium or blood cells.
Thus, there is currently no way to reverse permanent damage to the retina and restore vision. Drug treatments focus on treating the illness and its symptoms to prevent further damage to the retina. There is a need to reverse damage to the retina and restore vision by endogenously generating new retinal cells or transplanting retinal cells.
In tissues other than the eye, stem cells are used as a source for alternative treatments of disease or injury to tissues. Stem cells are undifferentiated cells that exist in many tissues of embryos and adult mammals. In embryos, blastocyst stem cells are the source of cells which differentiate to form the specialised tissues and organs of the developing fetus. In adults, specialised stem cells in individual tissues are the source of new cells which replace cells lost through cell death due to natural attrition, disease or injury. No stem cell is common to all tissues in adults. Rather, the term "stem cell" in adults describes different groups of cells in different tissues and organs with common characteristics.
Stem cells are capable of producing either new stem cells or cells called progenitor cells that differentiate to produce the specialised cells found in mammalian organs. Symmetric division occurs where one stem cell divides into two daughter stem cells. Asymmetric division occurs where one stem cell forms one new stem cell and one progenitor cell.
A progenitor cell differentiates to produce the mature specialized cells of mammalian organs. In contrast, stem cells never terminally differentiate (i.e. they never differentiate into a specialized tissue cells). Progenitor cells and stem cells are referred to collectively as "precursor cells". This term is used when it is unclear whether a researcher is dealing with stem cells or progenitor cells or both.
Progenitor cells may differentiate in a manner which is unipotential or multipotential. A unipotential progenitor cell is one which can form only one particular type of cell when it is terminally differentiated. A multipotential progenitor cell has the potential to differentiate to form more than one type of tissue cell. Which type of cell it ultimately becomes depends on conditions in the local environment as such as the presence or absence of particular peptide growth factors, cell-cell communication, amino acids and steroids. For example, it has been determined that the hematopoietic stem cells of the bone marrow produce all of the mature lymphocytes and erythrocytes present in fetuses and adult mammals. There are several well-studied progenitor cells produced by these stem cells, including three unipotenltial and one multipotential tissue cell. The multipotential progenitor cell may divide to form one of several types of differentiated cells depending on which hormones act upon it.
Weiss et al, Review, 1-13 (1996) summarises the five defining characteristics of stem cells as the ability to:
Thus, the key features of stem cells are that they are multipotential cells which are capable of long-term self-renewal over the lifetime of a mammal.
There is great potential for the use of stem cells as substrates for producing healthy tissue where pathological conditions have destroyed or damaged normal tissue. For example, stem cells may be used as a target for in vivo stimulation with growth factors or they may be used as a source of cells for transplantation.
There has been much effort to isolate stem cells and determine which peptide growth factors, hormones and other metabolites influence stem cell renewal and production of progenitor cells, which conditions control and influence the differentiation of progenitor cells into specialized tissue cells, and which conditions cause a multipotent progenitor cell to develop into a particular type of cell.
In several tissues, stem cells have been isolated and chlaracterised to develop new therapies to repair or replace damaged tissues. For example, stem cells have been isolated from the mammalian brain (Reynolds et al., Science 255:107 (1992)). WO 93/01275, WO 94/16718, WO 94/10292 and WO 94/09119 describe uses for these cells. WO 95/13364 reports that delivery of growth factors to the ventricles of the central nervous system ("CNS") stimulates neural stem cells to proliferate and produce progenitor cells which will develop into neurons, oligodendrocytes or astrocytes. All of these publications restrict the isolation or use of adult stem cells to the brain (in particular, the tissue around the brain ventricles, the subependyma, which is the remnant of the embryonic brain germinal zone). There is no reported isolation of a retinal stem cell from the adult peripheral nervous system ("PNS") of a mammal. There is no evidence for production of new neurons within the adult eye, so those knowledgeable about stem cells would not suspect that stem cells would be produced in the retina.
Stem cell cultures also provide useful assay cultures for toxicity testing or for drug development testing. Toxicity testing is done by culturing stem cells or cells differentiated from stem cells in a suitable medium and introducing a substance, such as a pharmaceutical or chemical, to the culture. The stem cells or differentiated cells are examined to determine if the substance has had an adverse effect on the culture. Drug development testing may be done by developing derivative cell lines, for example a pathogenic retinal cell line, which may be used to test the efficacy of new drugs. Affinity assays for new drugs may also be developed from the stem cells, differentiated cells or cell lines derived from the stem cells or differentiated cells.
The stem cells also provide a culture system from which genes, proteins and other metabolites involved in cell development can be isolated and identified. The composition of stem cells may be compared with that of progenitor cells and differentiated cells in order to determine the mechanisms and compounds which stimulate production of stem cells, progenitor cells or mature cells.
It would be useful if stem cells could be identified and isolated in areas of the CNS and PNS outside the adult brain, such as the retina. Medical treatments could then be developed using those stem cells. To date, no person has suggested that a retinal precursor cell in a mammal even exists beyond the embryonic stage of development, (a retinal stem cell would self-renew to exist from embryonic development to adulthood). There are retinal precursor cells in the embryonic eye which exhibit some of the characteristics of stem cells. However, since it is believed that these cells do not persist into the adult eye, this would indicate, by definition, that the embryonic precursors could only be retinal progenitor cells and not real retinal stem cells. The prior art teaches that it is highly unlikely that there is a retinal stem cell. Most of the prior art involves studies of embryonic precursor cells isolated from the mammalian eye (see e.g. Anchan et al., Neuron 6:923-936, (1991), Lillien et al., Development 115:253-266 (1992); Cepko et al., PNAS 93:589-595 (1996)). Precursor cells can be multipotential, however they usually have more restricted phenotype potential than stem cells. They also have only limited self-renewal capability. Anchan et al., Neuron 6:923-936, (1991), and Lillien et al., Development 115:253-266 (1992) both isolated embryonic retinal precursors in culture, however, they did not discuss retinal stem cells nor establish that retinal stem cells exist and can be isolated and purified. In summary, before this invention, no one expected that stem cells even existed in the retina or that the cardinal features of stem cells, self renewal and multipotentiality, could be found in those cells.
Current medical and surgical drug treatments are inadequate for restoring vision lost when retinal cells are damaged, so the potential clinical applications of pharmaceutical compounds containing retinal stem cells or to stimulate endogenous proliferation of retinal stem cells are tremendous. Retinal stem cells would have the potential to act as in vivo targets for stimulation by growth factors in order to produce healthy tissue. This may be done, for example, by injecting growth factors or genetically engineered cells which secrete growth factors into the eye. Some very preliminary work in this area was clone by Park et al., Developmental Biology 148:322-333 (1991). They stimulated retinal pigment epithelial cells in embryonic birds with FGF2 in vivo to regenerate a neural retina. However, this was in birds, not mammals, and only in embryonic birds. Moreover, the cells of the regenerated neuroretina formed in these chicks were not in their normal location. Thus, the photoreceptors, normally closest to the brain, were located farthest from it, and ganglion cells were closest to the brain. Thus, there is a clear need to develop techniques to safely and effectively target stem cells in vivo in mammals with growth factors in order to regenerate healthy eye tissue. The eye is easily accessible surgically or by injection, and it would be helpful if this accessibility could be exploited by targeting retinal stem cells in areas of damage.
It would also be useful if stem cells were discovered that could proliferate in the absence of growth factors.
A need also exists for a pharmaceutical composition containing retinal cells for transplantation in which (1) the composition is accepted by the patient, thus avoiding the difficulties associated with immunosuppression, (2) the composition is safe and effective, thus justifying the cost and effort associated with treatment, (3) the composition provides long term relief of the symptoms associated with the disease, (4) the composition is efficacious during and after transplantation. There is a clear need to develop retinal stem cell cultures which can act as a source of cells that are transplantable in vivo in order to replace damaged tissue.
There is also a need for retinal stem cell cultures or retinal cell cultures which may be used in toxicity testing, drug development and to isolate new genes and metabolites involved in cell differentiation. There is also a need for retinal cell cultures which may be used to develop derivative cell lines, such as retinoblastoma cell culture lines, for studying cancer or other diseases, disorders or abnormal states.